Systems and methods for identifying HLA-associated tumor peptides

Abstract

Systems and methods for determining amino acid sequences of peptides that bind to MHC-I or HLA-I complex or MHC-II or HLA-II complex are provided. One embodiment includes isolating peptides from MHC or HLA class I or class II-peptide complexes and adding one or more known labeled peptides of interest to form a sample containing labeled peptides and unlabeled isolated peptides. The method also includes analyzing the sample with an LC-MS/MS system to obtain sequence data of the peptides, and increasing the sensitivity of the LC-MS/MS system when the labeled peptide is detected by the LC-MS/MS system. The method then concludes with determining the amino acid sequence of the unlabeled peptides in an m/z range that includes the m/z of the labeled peptide. The system can be triggered to increase the sensitivity in or near the m/z of the labeled peptide using an algorithm or computer program.

The invention claimed is: 
     
       1. A method of determining amino acid sequences of peptides that bind to an MHC-I or HLA-I complex or an MHC-II or HLA-II complex, comprising:
 obtaining a sample comprising unlabeled isolated peptides from an MHC-I or HLA-I complex or an MHC-II or HLA-II complex; 
 adding one or more labeled peptides to the sample to form a mixed sample comprising the labeled peptides and the unlabeled isolated peptides; 
 continuously analyzing the mixed sample with an LC-MS/MS system to produce survey mass spectrometry scans of the sample, wherein detection of the one or more labeled peptides triggers a target label filter causing the system to scan isolated unlabeled peptides with a second mass spectrometer until the LC-MS/MS system does not detect the one or more labeled peptides. 
 
     
     
       2. A method of determining amino acid sequences of peptides that bind to an MHC-I or HLA-I complex or an MHC-II or HLA-II complex, comprising:
 obtaining a sample comprising unlabeled isolated peptides from an MHC-I or HLA-I complex or an MHC-II or HLA-II complex; 
 adding one or more labeled peptides to the sample to form a mixed sample comprising the labeled peptides and the unlabeled isolated peptides; 
 analyzing the mixed sample with an LC-MS/MS system to produce survey mass spectrometry scans of the sample; 
 integrating into the LC-MS/MS system a target label filter based on the survey mass spectrometry scans to monitor a list of the one or more labeled peptides, thereby creating an integrated LC-MS/MS system; 
 analyzing the mixed sample continuously with the integrated LC-MS/MS system, wherein detection of the one or more labeled peptides triggers the LC-MS/MS system to scan unlabeled isolated peptides until the integrated LC-MS/MS no longer detects the one or more labeled peptides; wherein the integrated LC-MS/MS system alternates between obtaining survey scans and scans of the one or more labeled peptides for the detection of endogenous peptides having the identical amino acid sequence of the one or more labeled peptides, respectively, and 
 
       determining the amino acid sequence of the unlabeled isolated peptides scanned by integrated LC-MS/MS system. 
     
     
       3. The method of  claim 1 , wherein the survey mass spectrometry scans have a duration of about 100 ms. 
     
     
       4. The method of  claim 1 , wherein the scan of the isolated unlabeled peptides has a duration of at least 400 ms. 
     
     
       5. The method of  claim 1 , wherein the filter causes the system to increase scan duration when the labeled peptides are detected. 
     
     
       6. The method of  claim 1 , wherein the obtaining step comprises isolating the unlabeled peptides from an MHC-I or HLA-I complex or an MHC-II or HLA-II complex. 
     
     
       7. The method of  claim 6 , wherein the isolating comprises affinity chromatography and/or column chromatography. 
     
     
       8. The method of  claim 6 , wherein the isolating comprises
 isolating by affinity chromatography the MHC-I or HLA-I complex or the MHC-II or HLA-II complex obtained from a tissue lysate; and 
 separating peptides from the complexes by column chromatography to produce the unlabeled isolated peptides. 
 
     
     
       9. The method of  claim 6 , wherein the isolating comprises
 passing a tissue lysate through an affinity chromatography column to produce a flow-through; 
 eluting the column to produce a first eluate, wherein the first eluate comprises MHC-peptide complexes and beta2M associated peptides; 
 passing the flow-through through a second affinity chromatography column to obtain a second eluate comprising MHC-peptide complexes and beta2M associated peptides; and 
 independently eluting the peptides from the first and second eluates to produce unlabeled isolated peptides. 
 
     
     
       10. The method of  claim 7 , wherein the affinity chromatography comprises an anti-HLA I column. 
     
     
       11. The method of  claim 7 , wherein the second affinity column comprises a pan HLA II column. 
     
     
       12. The method of  claim 7 , wherein the column chromatography comprises use of a C18 reverse phase chromatography column. 
     
     
       13. The method of  claim 1 , wherein the sample comprising unlabeled isolated peptides is a tissue lysate. 
     
     
       14. The method of  claim 13 , wherein the tissue lysate is produced from a tissue using a detergent. 
     
     
       15. The method of  claim 13 , wherein the tissue lysate is produced from prostate tissue, breast tissue, testicular tissue, thyroid, colon tissue, ovarian tissue, pancreatic tissue, nervous tissue, bone, bone marrow, peripheral blood mononuclear cells, metastatic tissue, cancer cell lines, biopsies, or ocular tissue. 
     
     
       16. The method of  claim 1 , further comprising determining an amino acid sequence of the unlabeled isolated peptides detected. 
     
     
       17. The method of  claim 1 , wherein a sequence of the unlabeled isolated peptides is determined in an m/z range that includes the m/z of the one or more labeled peptides. 
     
     
       18. The method of  claim 1 , wherein the labeled peptides are peptides labeled with heavy leucine, isoleucine, valine, threonine, lysine, tyrosine, phenylalanine, glycine, alanine, arginine, aspartic acid, or proline. 
     
     
       19. The method of  claim 17 , wherein the heavy leucine is (L)( 13 C 6   15 N 1 ). 
     
     
       20. The method of  claim 1 , wherein the method quantifies peptides present at a level of at least 30, 40, or 50 copies per cell.